This invention relates to nucleic acid sequences encoding novel modified xylose isomerases, to vectors carrying the sequences, to the polypeptides encoded by these sequences, to the production of a polypeptide encoded by these sequences and to the use of the isomerase in a process of ethanol production. The present invention further provides novel modified xylose isomerases that advantageously can be applied in the production of fructose syrups, in particular high fructose corn syrups.
D-xylose is a five-carbon sugar. It is present in nature, for example as xylan polymer in plant hemicellulose. As a carbon source, D-xylose has been reported to be utilised widely by bacteria and to a lesser extent by fungi. D-xylose is first converted to its keto isomer, D-xylulose, which is then phosphorylated to D-xylulose-5-phosphate, a normal pentose phosphate pathway intermediate.
There are two possible routes for the isomerisation of D-xylose: (a) direct conversion to D-xylulose by an isomerase; or (b) an oxidoreductive pathway, in which D-xylose is first reduced to xylitol and in a second reaction xylitol is oxidised to D-xylulose. The enzymes involved in this oxidoreductive pathway require coenzymes. The direct isomerisation is reported from bacteria and plant, while the oxidoreductive pathway is the main route for D-xylose to D-xylulose conversion in fungi.
Xylose isomerase catalyses the direct isomerisation of D-xylose to D-xylulose and vice versa, with the direction dependent on the relative concentration of the aldo and keto forms and the reaction conditions. Xylose isomerase also catalyses of interconversion of the 6carbon aldose sugar D-glucose and its keto isomer D-fructose. Thus, bacterial xylose isomerases are used industrially to produce D-fructose from D-glucose.
The economic use of lignocellulosic biomass as a renewable energy source is strongly dependent on the fermentation of xylose to ethanol. Since the best ethanol-producing organism, Saccharomyces cerevisiae is not able to utilise xylose, major efforts have been taken to develop recombinant strains possessing the necessary enzymes. The xylose reductase and xylitol dehydrogenase from Pichia stipitis were cloned and expressed in Saccharomyces cerevisiae. The resulting strains were able to ferment xylose, however the yields were far from the theoretical due to the xylitol excretion (Tantirungkij et al. (1993)).
Several attempts to circumvent this problem by introducing the alternative direct isomerisation pathway have been made. The xylose isomerases form two clusters based on the sequence homology, the actinomycetic genes showing less than 40% homology to the others. From the latter subgroup the Bacillus subtilis (Amore et al. (1989)) and the Escherichia coli (Sarthy et al. (1987)) xylose isomerases were expressed inactively in Saccharomyces cerevisiae, while introduction of the Clostridium thermosulfurogenes (Moes et al. (1996)) gene did not result in a protein that cross-reacted with a specific antibody. From the first subgroup the Actinoplanes missouriensis (Amore et al. (1989)) gene was cloned in yeast but the production of xylose isomerase failed due to an aberrant transcription start.
The Thermus thermophilus xylose isomerase only recently has been actively expressed in Saccharomyces cerevisiae (Walfridsson et al. (1996)). This gene is closely related to the actinomycetic genes showing approximately 65% homology. The enzyme, however, has only trace activity at 30xc2x0 C., because of its high (95xc2x0 C.) temperature optimum. The other important factor for the poor performance of the strain was the formation of xylitol, probably by the unspecific NADPH linked aldose reductase described by (Kuhn et al. (1995)), which is coded by the gene GRE3. The xylitol formation has a dual effect on the ethanol yield; it does not only lead to carbon loss, but it also competitively inhibits the xylose isomerase (Yamanaka (1969)). By increasing intracellular xylitol concentration the apparent affinity of the xylose isomerase towards xylose decreases and more xylose is channelled into xylitol, until the NADPH pool of the cell is depleted. In the presence of active xylose isomerase some xylitol can be formed also by the endogenous xylitol dehydrogenase described by (Richard et al. (1999)). To prevent the xylitol impairing the xylose isomerase and thereby blocking the xylose metabolism of the recombinant yeast, it is advantegous to have a xylose isomerase expressed which is less sensitive to the presence of xylitol.
In light of the previously described problems, there is a need to find a xylose isomerase being actively expressed in Saccharomyces cerevisiae and being more active at a broader range of pH values and temperatures.
In the present invention, a series of novel xylose isomerases have been isolated. They were modified from the Thermus thermophilus enzyme by random mutagenesis and identified as having elevated activity at mesophilic ( less than 40xc2x0 C.) temperatures, compared to the wild type enzyme. Expressed either in procaryotic or eucaryotic cells, the specific enzyme activity in the mutants is at least 10% higher than that of the wildtype enzyme. They are, therefore, expected to be more suitable for expression in yeast. The present invention thus comprises any isolated nucleic acid sequence which upon expression in a procaryotic or eucaryotic host cell codes for a polypeptide having at least 10% higher specific xylose isomerase activity, than the wild type xylose isomerase which nucleic acid sequence is shown in SEQ. ID. No. 2. The specific activity of the enzyme is to be determined as described in example 3, wherein one unit of xylose (glucose) isomerase activity is defined as the amount of crude enzyme needed to produce 1 xcexcmol of product per minute under the assay conditions. The specific activity (U minxe2x88x921 mgxe2x88x921) is herein determined from the activity and the protein concentration of the crude enzyme preparations.
The present invention further comprises any isolated nucleic acid sequence that is derived and shows at least one nucleic acid modification from the nucleic acid sequence shown in SEQ. ID. No. 2.
Furthermore, the mutants show a higher thermostability than the wildtype enzyme. One embodiment of the invention thus comprises an isolated nucleic acid sequence which upon expression in a procaryotic or eucaryotic host cell codes for a polypeptide having at least 10% higher specific xylose isomerase activity at temperatures above 80xc2x0 C. such as 81xc2x0 C., 82xc2x0 C., 83xc2x0 C., 84xc2x0 C., 85xc2x0 C., 86xc2x0 C., 87xc2x0 C., 88xc2x0 C., 89xc2x0 C., 90xc2x0 C., 91xc2x0 C., 92xc2x0 C., 93xc2x0 C., 94xc2x0 C., 95xc2x0 C., 96xc2x0 C., 97xc2x0 C., 98xc2x0 C., 99xc2x0 C. and/or 100xc2x0 C., than the wild type xylose isomerase which nucleic acid sequence is shown in SEQ. ID. No. 2.
In another embodiment of the invention, the mutants also show a higher acceptance for xylitol, which is an inhibitor to xylose isomerase, compared to the wildtype enzyme.
In a further embodiment of the invention, the mutated xylose isomerases have a broader pH range than the wildtype, even though it may have the same pH optimum at xcx9c7.0. The present invention therefor also comprises any isolated nucleic acid sequence which upon expression in a procaryotic or eucaryotic host cell codes for a polypeptide having at least 10% higher specific xylose isomerase activity at acidic pH values i.e. lower than pH 7.5 such as pH 7.4, pH 7.3, pH 7.2, pH 7.1, pH 7.0, pH 6.9, pH 6.8, pH 6.7, pH 6.6, pH 6.5, pH 6.4, pH 6.3, pH 6.2, pH 6.1, pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.5 and/or pH 4.0 than the wild type xylose isomerase which nucleic acid sequence is shown in SEQ. ID. No. 2.
In a preferred embodiment, the present invention provides:
An isolated nucleic acid sequence which codes upon expression in a procaryotic or eucaryotic host cell for a polypeptide having xylose isomerase activity at mesophilic temperatures, which nucleic acid sequence is selected from
a) nucleic acid sequences shown in SEQ. ID. No 1, wherein nnn at positions 1114-1116 stand for a codon coding for an amino acid other than glutamate, preferably a codon coding for glycine and nnn at positions 487-489 stand for a codon coding for any amino acids, preferably phenylalanine, more preferably leucine
b) the complementary strand of the sequence defined in (a) above;
c) nucleic add sequences which hybridise to the sequences defined in (a) or (b) above and the residues corresponding to the residues nnn and nnn in SEQ. ID. No 1 are as defined in (a) or (b) above;
d) nucleic acid sequences which, but for the degeneracy of the genetic code, would hybridise to the sequences defined in (a), (b) or (c) above and which code for the same polypeptide as those defined in (a), (b) or (c) above;
a polypeptide encoded by a nucleic acid sequence as defined above;
a vector comprising a nucleic acid sequence as defined above;
cells transformed or transfected with such a vector; a process of producing ethanol which comprises:
(i) contacting such transformed cells with a substrate that contains one or more carbon sources selected from xylose and polymerised xylose moieties;
(ii) culturing the said cells in conditions under which the isomerisation of D-xylose to D-xylulose occurs and under which the D-xylulose is further catabolised to ethanol; and
(iii) recovering the ethanol;
a process of producing a polypeptide as defined above comprising expressing a nucleic acid sequence as defined above in a cell as defined above;
a process of producing D-fructose which comprises:
(i) contacting such polypeptide with a substrate containing D-glucose;
(ii) incubating the substrate with the polypeptide in conditions under which the isomerisation of D-glucose to D-fructose occurs;
(iii) recovering the mixture of D-glucose and D-fructose
The nucleic acid sequences of the present invention are preferably DNA, though they may be RNA or even PNA. It will be obvious to those of skill in the art that, in RNA sequences according to the invention, the T residues shown in SEQ. ID. No. 1 will be replaced by U.
The nucleic acid sequences of the present invention include in the sequence shown in SEQ. ID. No. 1, wherein the nnn at the positions 1114-1116 stand for a nucleotide triplet, which upon expression codes for an amino acid other than glutamate, preferably glycine. Such nucleotide triplet is for example GGA. Similarly, the nnn at the positions 487-489 stand for a nucleotide triplet, which upon expression codes for any amino acid, preferably phenilalanine, more preferably leucine. Such a nucleotide triplet is for example CTC.
As apparent from the ove, the nucleic acid sequences of the present invention are not limited to the sequences of SEQ. ID. No 1. Rather, the sequences of the invention include sequences that are closely related to these sequences and that encode a polypeptide having xylose isomerase activity, xylose isomerase activity being the ability to catalyze the direct interconversion of xylose to xylulose. For example, four preferred sequences of such polynucleotides are shown in SEQ. ID. No. 3, 5, 7, and 9. These nucleic acid sequences may be prepared by altering SEQ. ID. No. 1 by any conventional method, or they may be isolated from an organism or made synthetically. Such alterations, isolations, or syntheses may be performed by any suitable method, for example by the methods of Sambrook et al. (Sambrook et al. (1989)).
For example, the sequences of the invention include sequences that are capable of selective hybridisation to those of SEQ. ID. No. 1; preferably those wherein the residues selectively hybridising to the residues marked as nnn in SEQ. ID. No. 1 are coding for an amino acid other than glutamate, preferably glycine; and which code for a polypeptide having xylose isomerase activity. Such hybridisation may be carried out under any suitable conditions known in the art (see Sambrook et al. (1989)). xe2x80x9cHybridisationxe2x80x9d is a process in which a strand of nucleic acids joins with a complementary strand through base pairing. The degree of complementarity of two non-identical, but very similar strands, and their length influence the hybridisation conditions. In the present invention, the conditions for hybridisation are meant to be those of a stringent hybrisisation according to (Sambrook et al. (1989)).
Also the sequences of the invention include sequences that are different from those defined above because of the degeneracy of the genetic code and encode the same polypeptide. Degenerate code stands for a genetic code in which a particular amino acid can be coded by two or more different codons. Degeneracy occurs because of the fact that of the 64 possible base triplets, 3 are used to code the stop signals, and the other 61 are left to code for only 20 different amino acids.
Terms and abbreviations in this document have their normal meanings unless otherwise stated. All nucleic acid sequences are written in the direction from the 5xe2x80x2(stands for prime) end to the 3xe2x80x2 end also referred to as 5xe2x80x2 to 3xe2x80x2. All amino acid or protein sequences, unless otherwise designated, are written from the amino terminus (N-terminus) to the carboxy terminus (C-terminus).
The codon usage is the standard published and internationally used single or triple letter amino acid code.
The word xe2x80x9cbase pairxe2x80x9d or xe2x80x9cbpxe2x80x9d refers to DNA or RNA. The single letters A, C, T and G correspond to the 5-monophosphate forms of the deoxyribonucleosides (deoxy)adenine, (deoxy)cytidine, (deoxy)guanine and (deoxy)thymidine, respectively. Furthermore, the abbreviations U, C, G and T may correspond to the 5xe2x80x2-monophosphate forms of the ribonucleosides uracil, cytidine, guanine and thymine, respectively, occurring in RNA molecules. xe2x80x9cBase pairxe2x80x9d in double stranded DNA refers to a pairing of A with T or C with G. In a heteroduplex of RNA paired with DNA, base pair may refer to a pairing of A with U or C with G.
The term xe2x80x9cvectorzxe2x80x9d stands for a nucleic acid compound used for the transformation of cells. A vector contains a polynucleotide sequence corresponding to appropriate protein molecules which, when combined with appropriate control sequences, confer specific properties on the host cell to be transformed. Plasmids, viruses and bacteriophage are suitable vectors. Artificial vectors can be constructed by cutting and joining DNA molecules from different sources using restriction enzymes and ligases. The term vector also includes recombinant DNA cloning vectors and recombinant DNA expression vectors.
The term xe2x80x9cPromoterxe2x80x9d stands for a DNA sequence which directs transcription of DNA to RNA,
The term xe2x80x9cplasmidxe2x80x9d refers to an extrachromosomal (usually) self-replicating genetic element. Plasmids are commonly designated by a lower case xe2x80x9cpxe2x80x9d followed by letters or numbers. The plasmids used here are either commercially available, publicly available on an unrestricted basis or can be constructed from available plasmids following published procedures. In addition, equivalent plasmids to those described are known and are apparent to the skilled worker in this field.
xe2x80x9cTransformationxe2x80x9d means the introduction of DNA into an organism in a way that it can replicate itself, either as an extrachromosomal element or by chromosomal integration. Methods of transforming bacterial and eukaryotic hosts are well known. Many of these methods, such as nuclear injection, protoplast fusion or calcium treatment with calcium chloride are summarized in (Sambrook, et al. (1989)).
xe2x80x9cTransfectionxe2x80x9d is the operation of adding an expression vector to a host cell, whereby the cell itself takes up the DNA molecule and either integrates it into the chromosomes (stable transfection) or keeps it separate from the chromosomal DNA in the nucleus as a transient transcribed plasmid (transient transfection). Many different methods of transfection are known, for example calcium phosphate co-precipitation and electroporation. A successful transfection can be monitored by a specific site introduced in the transfected DNA molecule which can be recognised by an antibody. This DNA site can either be a specifically introduced xe2x80x9cflagxe2x80x9d or xe2x80x9ctagxe2x80x9d meaning a specific sequence which is easily recognised by an antibody. It could also be an intrinsic part of the expressed gene, recognised by a specific antibody. Another possibility would be the fusion to a fluorescent protein like eg GFP (green fluorescent protein).
The expressions xe2x80x9ccomplementaryxe2x80x9d and xe2x80x9ccomplementarityxe2x80x9d refer to the matter of base pairing of purines and pyrimidines that associate through hydrogen bonding in double stranded nucleic acid. The base pairs guanine and cytosine, adenine and thymidine as well as adenine and uracil are complementary.
xe2x80x9cIsolated amino acid sequencexe2x80x9d refers to any amino acid sequence, constructed or synthesised, which is locationally distinct from the naturally occurring sequence.
A xe2x80x9cprimerxe2x80x9d is a nucleic acid fragment, which functions as an initiating substrate for enzymatic or synthetic elongation.
The term xe2x80x9ca polypeptidexe2x80x9d in the present application is generally understood to mean a polypeptide of the invention, as shown for example in SEQ. ID. No. 4, 6, 8, or 10. It is also within the meaning of xe2x80x9ca polypeptidexe2x80x9d that several polypeptides can be used, i.e. in the present context xe2x80x9caxe2x80x9d means xe2x80x9cat least onexe2x80x9d unless explicitly indicated otherwise.
The sequence xe2x80x9cidentityxe2x80x9d can be calculated as                     (                              N            ref                    -                      N            dif                          )            ⁢      100              N      ref        ,
wherein Ndif is the total number of non-identical residues in the two sequences when aligned, and wherein Nref is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCMTC (Ndif=2 and Nref=8). A gap is counted as non-identity of the specific residue(s), ie the DNA sequence AGTGTC will have a sequence identity of 75% with the DNA sequence AGTCAGTC (Ndif=2 and Nref=8). Sequence identity can alternatively be calculated by the BLAST program, eg the BLASTP program (Pearson W. R and D. J. Lipman (1988))(www.ncbi.nlm.nih.gov/cgi-bin/BLAST). Alignment can be performed with the global align algorithm with default parameters as described by (Huang and Miller (1991)), available at http://www.ch.embnet.org/software/LALIGN_form.html.
One preferred embodiment of the invention thus comprises a nucleic acid sequence, which nucleic acid sequence is identical at least 85%, such as at least 90%, at least 95%, at least 99% or at least 100% to any of the sequences defined above.
A yeast expressing active xylose isomerase can be used in a process for producing ethanol from xylose containing materials. Suitable substrates for ethanol production include xylose itself and any compound that the cells can convert into xylose. For example, suitable substrates include polymers that contain xylose moieties. Thus, xylan, arabinoxylan and xyloglucan polymers, which comprise xylose moieties, are suitable substrates. Accordingly, hemicellulosic and lignocellulosic substrates, such as plant biomass, are suitable substrates.
In processes of ethanol production according to the present invention, xylose is typically released from xylan by enzymatic or chemical hydrolysis under acidic or basic conditions, or by heating or by a combination of these techniques. For example xylose can be released from xylan by a combination of acidic hydrolysis and heating under pressure. The yeasts typically used in ethanol production are not capable of enzymatically hydrolysing xylans to release xylose, although some yeasts are capable of doing so and of metabolising the xylose to ethanol. Such yeasts, when transformed with a vector according to the invention, are included within the scope of the invention.
Although the preferred processes of ethanol production according to the invention comprise contacting transformed or transfected cells with a suitable substrate, other processes of producing ethanol are also possible. For example, a polypeptide according to the invention may be added to a cellular fermentation broth in order to liberate xylose outside the cells, which is then taken up and metabolised by them.
In the present invention, ethanol will typically be produced by a fermentation broth that comprises yeast cells according to the invention, water, one or more sources of xylose and other nutrients. These nutrients preferably include a suitable nitrogen source, such as (NH4)2SO4. The presence of hexose sugars, such as glucose may enhance both ethanol formation productivity and growth. Also, the presence of Mn2 or Mg2 ions is desirable as one of these ions is necessary to the function of the mutant xylose isomerases. Further, the presence of low levels of oxygen is desirable, especially in fermentation broths containing Saccharomyces cerevisiae. 
The process may be carried out at any temperature that facilities ethanol production, but temperatures from 15 to 40xc2x0 C. are preferred and temperatures from 30 to 35xc2x0 C. are particularly preferred. In one embodiment of the present invention, the novel xylose isomerases therefor show at least 10% elevated activity at temperatures ranging from 20-60xc2x0 C., such as in an especially preferred embodiment at 30-35xc2x0 C.
For the ethanol-producing processes of the invention, it is preferred that transformed or transfected yeast cells, especially those of S. cerevisiae are used. Recently a Saccharomyces cerevisiae strain was constructed with the GRE3 gene disrupted (WO99/54477). This strain shows reduced levels of xylitol formation, when it is metabolising xylose. In one preferred embodiment of the invention, cells of a S. cerevisiae strain according to the patent application (WO99/54477) are used. As the person skilled in the art will be aware of, though, this should not exclude the possible usage of other transfected yeast cells, such as a cell of a species selected from a list that should be regarded only as illustrative and not exclusive, consisting of Pichia sp., Candida sp., Shizosaccharomyces sp., Zygosaccharomyces sp. and Saccharomyces sp. Furthermore, other Saccharomyces families are embodied in the scope of the invention, for example; Saccharomyces cerevisisae, Saccharomyces bayanus and Saccharomyces carlsbergensis. 
In the ethanol-producing processes of the invention, the substrate is wholly or partly converted to ethanol, which may be recovered by any suitable means known in the art. The xylose isomerases according to the invention are also suitable for industrial high fructose syrup production. In this process D-glucose, preferably in the form of starch hydrolysate, is partially converted to D-fructose. Typical process conditions are 60xc2x0 C., pH 7.5 with 45 ppm Mg2+ and 45% w/w D-glucose as substrate. The substrate is preferably obtained as starch hydrolysate, produced for example from wheat starch with the methods described in (Godfrey and Reichelt (1983)).
In a conventional industrial process, the aim is, in most cases, to obtain a sugar solution, in which 40 to 45% of the sugar is fructose. When the activity of the enzyme decreases with the ageing of the column, the desired level is maintained by reducing the flow rate.
Random Mutagenesis of the xylA Gene
The xylA gene of the Thermus thermophilus was amplified using polymerase chain reaction (PCR) from the pBXI plasmid (Walfridsson et at (1996)) as template. Three samples with the following (identical) composition were prepared:
The nucleic acid sequence of Primer 1 is shown in SEQ. ID. No.11, that of Primer 2 is shown in SEQ. ID. No. 12.
The samples were placed in a thermocycler (Perkin Elmer GeneAmp2400) and subjected to the following temperature program, where the 2nd segment was repeated 6 times:
Temperature program:
The samples were mixed and divided again into three aliquots to equalise the concentrations. The following reagents were added to these samples:
The temperature program described above was performed in the thermocycler mentioned above with a single 2.segment. Half of the samples (50 xcexcl) was removed and saved on xe2x88x9220xc2x0 C. (samples A1, B1 and C1). To the remaining 50 xcexcl samples the following reagents were added:
The temperature program described above was performed in the thermocycler mentioned above with only one 2.segment. Half of the samples (50 xcexcl) was removed and saved on xe2x88x9220xc2x0 C. (samples A2, B2 and C2).
With the remaining 50 xcexcl samples the addition of the reagents and the temperature program were performed again with the same conditions as above. 50 xcexcl of each sample were saved on xe2x88x9220xc2x0 C. (A3, B3 and C3). The rest of them were applied to agarose (2%) gel electrophoresis to check the reaction efficiency.
All of the nine samples (A1-3, B1-3, and C1-3) were purified with High Pure(trademark) PCR Product Purification Kit (Roche Diagnostics). With each samples the following reaction were set up:
The temperature program described above was performed in the thermocycler mentioned above with ten repeated 2.segment. The samples were purified as before and used for ligation experiments.
Cloning and Selection of Mutant xylA Genes
The mutant xylA genes obtained in the previous example were inserted into the general cloning vector pGEM-t Easy (Promega Corp.). 300 ng of vector (pGEM-t Easy) was incubated overnight at 4xc2x0 C. with 200 to 500 ng of insert in total volume of 10 xcexcl. A control reaction without insert was also conducted.
The ligation products were transformed into E. coli HB101 (xylA5), which is a xylose isomerase deficient strain. HB101 High Efficiency Competent Cells (Promega Corp.) were transformed according to the suppliers recommendations, and 3xc3x97100 xcexcl or 5xc3x97200 xcexcl were plated on LB-agar supplemented with ampicillin (50 xcexcl/xcexcl) and X-gal (20 xcexcg/xcexcl). Colonies where moved to MacConkey-agar plates with ampicillin and xylose (2%), either with toothpicks or by replica plating. The plates were incubated at 37xc2x0 C. overnight and thereafter incubated at different temperatures (37xc2x0 C. and 55xc2x0 C.) until the colour reaction, which indicates xylose utilisation (orange to red), was seen. On the MacConkey plates, E. coli DH5xcex1 (harbouring pUC18 for ampR) and E. coli HB101 (harbouring pGEM-t Easy for ampR) were used as positive and negative control, respectively. Note that the strain HB101 has Xylxe2x88x92, whereas the strain DH5xcex1 has Xyl+ phenotype.
All colonies similar colour reaction (xylose utilisation) to the positive control were spread on new MacConkey-amp-xylose plates, LB-amp plates and inoculated in liquid LB-amp (50 xcexc/xcexcl). Plasmid DNA from the liquid cultures were prepared using the CTAB method, and subjected to EcoRI digestion to establish if they contained the xy/A-insert. Plasmids with insert were then retransformed into E. coli HB101 to confirm their phenotype on MacConkey plates. Correct colonies were cultured as above and the plasmids purified with Nucleon mini plasmid DNA preparation kit (Amersham). These DNAs were used in the subsequent experiments and to determine the nucleic acid sequences of the mutated genes. Schematic representation one of the obtained plasmid is shown in FIG. 1.
Kinetic Characterisation of the Mutant Xylose Isomerases
E. coli HB101 haboring plods PUSM1010, PUSM1021, pUSM1024, pTJSM1026, and pUSMwt, with the mutated and the wildtype xylose isomerase genes respectively (as described in the previous two examples) were grown at 37xc2x0 C. in 50 ml LB media containing 100 xcexcl/ml ampicillin. The cells were harvested by centrifugation in the stationary phase of growth and washed once with ice cold distilled water. Washed cells were resusprended in 100 mM triethanolamine, pH 7.0, 0.5 mg/ml lysozyme (130000 U/mg), 0.25 mg/ml DNase, and 1 mM phenylmethylsulphonyl fluoride (PMSF) in dimethylsulfoxide (DMSO). The suspensions were kept at room temperature for one hour and then on ice for two hours before being put in a freezer at xe2x88x9220xc2x0 C. The cell lysates were thawed on ice, cell debris was removed by centrifugation (1300 rpm for 15 minutes at 4xc2x0 C.) and the supernatants were used as the crude enzyme preparations.
Protein concentration was determined with the Bradford method (Bradford (1976)) using the Pierce protein reagent with bovine serum albumin as standard.
The xylose isomerase was assayed by incubating 700 mM D-xylose with suitable amount of crude enzyme preparation in the presence of 10 mM MnCl2 and 100 mM triethanolamine, pH 7.0 at the desired temperature in 0.5 ml volume. Glucose isomerase activity was assayed under the same reaction conditions, except that D-glucose instead of xylose was used in the reaction mixture. The reaction was terminated after 10 minutes by adding 150 xcexcl 50% trichloroaceticacid. The reaction mixture was neutralized with 185 xcexcl 2 M Na2CO3. The formed xylulose or fructose was measured on a COBAS MIRA (Roche) automated spectrophotometer via enzymatic reduction with 0.04 or 0.5 units of Sorbitol Dehydrogenase (purchased from Roche), respectively in the presence of 0.15 mM NADH at pH 7.0, 37xc2x0 C. in 200 xcexcl reaction volume. The rate of disappearance of NADH was followed at 340 nm and the concentration of D-xylulose or D-fructose was calculated using calibration curves. One unit of xylose (glucose) isomerase activity was defined as the amount of crude enzyme needed to produce 1 xcexcmol of product per minute under the assay conditions. The specific activity (U minxe2x88x921 mgxe2x88x921) was determined from the activity and the protein concentration of the crude enzyme preparations. Comparison between the specific activities at 30xc2x0 C. is shown in FIG. 2 and Table 1. The change of the specific activity with the temperature for each enzyme is displayed in FIG. 3. The temperature optimum for the mutants were roughly the same as for the wild type enzyme except for mutant 1021, which had xcx9c10xc2x0 C. lower temperature optimum.
The kinetic parameters Vmax (xcexcmol minxe2x88x921 mgxe2x88x921) and Km (mM) were determined from Michaelis-Menten plots of specific activities at various substrate concentrations by nonlinear regression analysis with Sigma Plot (SSPS Inc.) software. Typically, duplicate measurements were used to determine the Km value at 6-10 concentrations of the substrate, with a concentration range covering the value of Km.
The inhibition constant for xylitol (Ki) was determined by incubating the crude enzyme preparations with different xylose concentrations (20-400 mM) at different fixed xylitol concentrations. The mutants had a higher Ki for xylitol, which is an inhibitor to xylose isomerase, compared to the wildtype enzyme.
The effect of pH on the activity of the enzymes was followed in the pH range 5-10 in a buffer prepared by mixing acetate, piperazine-N,Nxe2x80x2-bis-(2-ethanolsulphonic acid) (PIPES), N-2-hydroxyehylpiperazine-Nxe2x80x2-2-etanesulfnicacid (HEPES) and glycine at final concentration 50 mM each as described by (Dekker at al. (1992) Appl. Mirobiol.
Biotechnol. 36: 727-732). The pH was adjusted at 60xc2x0 C. with NaOH. The assay was performed at 60xc2x0 C. in mixture containing 700 mM D-xylose 10 mM MnCl2. Above pH 7.0 corrections were made for the chemical isomerisation of D-xylose. The change of the specific activity with the pH for each enzyme is shown in FIG. 4. The mutated xylose isomerases all had a broader pH range than the wildtype, but the same pH optimum xcx9c7.0. Thermal stability of wild type xylose isomerases and mutated xylose isomerases where tested by incubating metal free enzyme preparations at 70xc2x0 C. in 200 mM triethanolamine, pH7.0, 10 mM MnCl2. Residual activities of timed aliquots were recorded using xylose as substrate.
Table 2 illustrates a summary of the catalytic properties of the tested mutated xylose isomerases and the wild type xylose isomerase.
Furthermore, the effect of different bivalent cations (10 mM) on the activity of EDTA treated enzymes was investigated. The specific activity with 10 mM MnCl2 at 60xc2x0 C. was set to 100% for each enzyme. The results are shown in table 3.
Construction of Yeast Expression Vector
The vector pMA91 (Mellor et al. (1983)) was hydrolysed with the restriction enzyme HindIII (Roche) to obtain the yeast PGK1 promoter and terminator sequences. The digested DNA was electrophorised on 1.0 % agarose gel and the 1.8 kb fragment was cut out from the gel with a scalpel. The DNA fragment was recovered from the agarose slice with the QIAqiuck Gel extraction kit (QIAGEN). YEplac112 (Gietz and Sugino (1988)) was linearised with HindIII, digested with Shrimp Alkaline Phosphatase (Roche) and purified with QlAquick PCR purification kit (QIAGEN). The two DNA fragments were ligated using T4 DNA ligase (Gibco BRL) according to the supplier""s recommendations. Ligation products were transformed into E. coli HB101 and plated onto LB agar plates with 100 xcexcg/ml ampicillin. The plates were incubated overnight on 37xc2x0 C., 5 colonies were picked up and inoculated into 5 ml LB media with 100 xcexcg/ml ampicillin. The cultures were incubated overnight on 37xc2x0 C. with rocking. Plasmids were isolated with QIAprep Spin kit (QIAGEN) and used for restriction analysis. One isolate showing the correct HindIII digestion pattern was chosen and it was inoculated into 100 ml LB media with 100 xcexcg/ml ampicillin. The culture was grown overnight on 37xc2x0 C. with 200 rpm shaking. Large scale plasmid preparation was performed with QIAGEN MAXIPREP (QIAGEN) and the obtained plasmid (in the following referred as YEplacPGK) was used in the following experiment. pUSM1026 was transformed into E coli GM2163 (damxe2x88x92) and plated onto LB agar with 100 xcexcl/ml ampicillin and 10 xcexcg/ml chloramphenicol. A colony was inoculated into 5-10 ml LB with 100 xcexcg/ml ampicillin and 10 xcexcg/ml chloramphenicol. The culture was grown overnight on 37xc2x0 C. with rocking. Unmethylated plasmid was isolated from the culture with Quantum Prep plasmid miniprep kit (BIO-RAD). The plasmid was cut with BclI (Roche) on 50xc2x0 C. and applied to a 1.0% agarose gel. The 1.2 kb fragment was cleaved out with a scalpel and purified from the agarose gel with QlAquick Gel extraction kit (QIAGEN). 1-5 xcexcg of YEplacPGK was digested with BglII (Roche), dephosphorilated with Shrimp Alkaline Phosphatase (Roche) and purified with QlAquick PCR purification kit (QIAGEN). The two DNA fragments were mixed and ligated with T4 DNA ligase (Gibco BRL) under the conditions recommended by the manufacturer. The ligation mixture was transformed into E. coli and plated onto LBagar with 100 xcexcg/ml ampicillin. 8-10 colonies were picked and inoculated into 5-10 ml LB+100 xcexcg/ml ampicillin. After the cultures had grown overnight on 37xc2x0 C. with rocking, plasmids were isolated with Quantum Prep plasmid miniprep (BIO-RAD). The plasmids were sequenced to determine the direction of the insert compared to the promoter. One preparatum with the correct sequence was chosen to be used in the subsequent experiments and named M1026.
To construct a control plasmid similar to the one above, but containing the wild type xylA gene, the plasmid pBXI (Walfridsson et al. (1986)) was partially hydrolysed with HindIII (Roche). After separating the fragments on 0.7% agarose gel the 3.0 kb fragment was cut out and purified with QlAquick Gel extraction kit (QIAGEN). The fragment carrying the promoter-gene-terminator cassette was inserted into YEplac112, which was linearised with HindIII and dephosphorilated before. The ligation products were transformed into E. coli and plasmids were reisolated from 3 colonies, as described above. An isolate showing the expected restriction pattern with HindIII was used in the following experiments and named YEplacTthXI. The constructed yeast expression plasmids are shown in FIG. 5 (YEplacPGK, YEplacM1026) and FIG. 6. (YEplacTthXI).
Expression of the Mutant xylA in Saccharomyces cerevisiae 
Plasmids YEplacPGK, YEplacM1026 and YEplacTthXI were transformed into Saccharomyces cerevisiae CEN.PK2-1D with the method of (Gietz and Woods (1994)). The transformants were plated onto SC-Trp plates and incubated on 30xc2x0 C. for 2 days. Transformants were inoculated into 5 ml SC-Trp media and were grown overnight on 30xc2x0 C. with rocking. The cells were harvested by centrifugation (10 minutes, 5000 g), suspended in distilled water and centrifuged again. The cell pellets were suspended in 500 xcexcl Y-PER reagent (Pierce) supplemented with 1 mM phenylmethylsulfonyl fluoride. The extraction was performed on room temperature for 30 minutes with shaking. After removing the cell debris by centrifugation for 5 minutes at 13000 g, the resulting supernatants were used for determining the xylose isomerase activity. The xylose isomerase assay and the protein concentration measurements were as described in Example 3.